Structure-Function Relationship of the Chloroplastic Glutaredoxin S12 with an Atypical WCSYS Active Site

Abstract

Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol 28 WCSYS 32 active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys 87 ) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys 87 exhibits a certain reactivity, since its pK a is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys 29 ) and that the enzyme does not form inter-or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation.

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

“…2). This result was unexpected as AtGrxC5 possesses the active site Trp residue, which was thought previously to prevent the incorporation of an Fe-S cluster in the close paralog PtGrxS12 (27).…”

“…GrxS12, -S14, and -S16 in poplar as well as GrxS14 in A. thaliana (previously referred to as AtGRXcp) have already been confirmed as being located in plastids by GFP fusion (20,27,44). From localization prediction programs, only three additional A. thaliana Grxs exhibit a potential plastidial targeting sequence, namely AtGrxC5 (class I), AtGrxC10, and AtGrxS13 (class III) (10,11).…”

“…Protein purity was checked by SDS-PAGE, and protein concentrations were determined spectrophotometrically using a molar extinction coefficient at 280 nm of 8 for PtGrxS12 S32C. PtGrxC1, PtGrxC3, PtGrxS12, PtTrxh1, AtMsrB1, and AtPrxIIE were purified as described previously (8,14,27,30,31).…”

“…A similar ligation was observed for E. coli Grx4, a class II Grx, but the different orientation of the two monomers was proposed to be responsible for the differential cluster lability (26). The capacity of class I Grxs to bind an Fe-S cluster has been studied in detail by site-directed mutagenesis, and it was proposed that the nature of the residues adjacent to the first active site Cys determined this capacity (8,27). In particular, for Grxs with a YCPFC or YCPYC active site, the replacement of the proline by a glycine, simulating GrxC1 (YCGYC), was sufficient to allow the incorporation of such a cluster (8) In addition, it has been hypothesized that the Trp residue adjacent to the catalytic cysteine residue in GrxS12 (WCSYS active site) prevented cluster incorporation because changing it into a Tyr allowed cluster incorporation (27).…”

Enzyme structure captures four cysteines aligned for disulfide relay

“…In addition, the NAH group of Phe121 is in position to form a hydrogen bond with the carbonyl group of the residue preceding the Erv domain C A cysteine (Gly451 C@O). These interactions can be compared with other structures of mixed disulfides involving a CXXC motif, such as complexes of glutaredoxin with glutathione, 19 thioredoxin and a target protein, 20 and the bacterial periplasmic dithiol/disulfide oxidoreductases DsbA and DsbB. 8 In each of these cases, the substrate peptide or target region is positioned antiparallel to the loop containing the cis-proline, and the NAH and C@O groups of the substrate cysteine backbone are hydrogen bonded to the two backbone amide groups immediately upstream of the proline [ Fig.…”